Development of fungal expression plasmids to facilitate promoter profiling and gene silencing studies in Coprinopsis cinerea /

Abstract

The ink cap mushroom Coprinopsis cinerea (formally Coprinus cinereus) has long been regarded as one of the best model systems for the study of basidiomycete fungi. Most attention in C. cinerea has been focused on processes such as mating type determination and on meiosis. Little detail is known about the other aspects of the biology of this fungus, such as the cellular mechanisms involved in the development of fruiting structures. This research describes the development of fungal expression plasmids to facilitate promoter profiling and gene silencing studies in C. cinerea.

The main focus of this research was the development of expression cassettes to study the C. cinerea serine protease enzymes (SPRs). Serine proteases have been shown to be significant in both post-harvest spoilage of mushrooms and nutrient acquisition from compost. These expression cassettes will enable the elucidation of the role these enzymes play in mushroom fruiting body development and nutrient acquisition.

Initial work focused on the development of a rapid DNA extraction method for fungal samples which was central to this work. A protocol using the Precellys Bead Mill Homogeniser was successfully developed and a technical report was produced and published online, which is available at: https://homogenizers.net/pages/p-dna-extractionfrom-fungal-organisms.

Early stages in the construction of expression cassettes included the identification, isolation and analysis of the promoters of the serine protease genes and of selected fruiting body development genes (Agaricus bisporus lectin (abl), C. cinerea ornithine transferase (oat), and C. cinerea galectin genes (cgl1 and cgl2). In particular, work focused on the development of a GFP-promoter fusion plasmid containing the C. cinerea serine protease 10592 promoter. The plasmid was designed and constructed to link the promoter to efficient Green Fluorescent Protein (eGFP) producing the peGFPi10592 construct. The construction of this vector is a significant addition to the C. cinerea molecular toolkit. In addition to this, GFP fusion cassettes were also designed for the SPR promoters 04562 and 10615. The results presented here include work undertaken to identify and isolate the C. cinerea SPR promoter regions of 04562 and 10615 and the preparation of plasmid backbones for cloning these fragments. Antisense cassettes were also designed for the SPR genes 10592 and 04562. The results seen here illustrate the successful isolation of these genes and the generation of the corresponding plasmid backbones ready for cloning experiments. This research will form a foundation for future experiments to study C. cinerea SPRs and fruiting body development genes in the fungal life cycle.